Oral candidiasis (OC, caused by Candida) is a major complication in immunosuppressed patients, including those infected with HIV, cancer, and diabetes, as well as infants. One of the risk factors for OC is the use of antibiotics, which is known to alter the microbial flora. Interactions between Candida and members of the oral microbiome have not been investigated. We performed preliminary studies to: (a) characterize the oral bacterial microbiome (bacteriome) and oral fungal microbiome (mycobiome), and (b) determine whether changes in bacteriome/mycobiome affect Candida colonization. We showed: (1) core oral bacteriome (COB) comprises 14 genera in HIV-infected and uninfected individuals, of which 13 were common, (2) core oral mycobiome (COM) comprised 5 genera (of which Candida and Penicillium were shared between the two cohorts), (3) deceased abundance of the yeast Pichia coincided with increase in Candida abundance, suggesting an antagonistic association between these two fungi. To confirm this antagonistic interaction, we performed additional studies and showed that: (4) Pichia spent media (PSM) inhibited Candida growth, adherence, germination, and its ability to form biofilms, (5) this anti-Candida activity was Pichia specific. (6) PSM exhibited broad antifungal activity inhibiting Cryptococcus, Aspergillus and Fusarium as well. Next, (7) used a murine model of OC and showed that PSM-treatment reduced Candida infection in vivo, as shown by reduction in tongue fungal load and histological evaluation. (8) Treatment of PSM with proteinase-K abrogated its anti-biofilm activity, while metabolites extracted from PSM had no effect on Candida, showing that the active component/s of PSM is proteinaceous in nature. Next, (9) we performed proteomics analysis of PSM and found 13 proteins with a high match score (indicating strong identity match), including 6 glycolytic enzymes, 3 transcription factors, a plasma-membrane ATPase, a glucanase, and a serine proteinase. Since, glucanase and proteinase were shown to mediate the inhibitory activity of Pichia against plant fungi we hypothesize that the anti-Candida activity of PSM is mediated by these proteins. Finally, (10) exposure to pepstatin A (proteinase inhibitor) abrogated the anti-Candida activity of PSM implicating proteinase in PSM inhibition. In this application, we will purify and characterize the glucanase/proteinase proteins, determine their mechanism/s of action, and validate their efficacy using a murine model of OC. The specific aims of the current proposal are: Aim 1. Purify and characterize glucanase and proteinase proteins of PSM. Aim 2. Determine the mechanism/s by which Pichia glucanase/proteinase inhibit Candida. Aim 3. Determine the efficacy of Pichia glucanase/proteinase in vivo. Understanding the mechanism/s by which Pichia proteins inhibit these organisms is critical in providing insight into the pathogenesis and control of fungal infections.